The present invention relates to methods for measuring and analyzing the synthesis and turnover of polymers within cells or tissues, or during chemical reactions outside of the cell, using components of the polymer containing stable isotopes as probes.
Living systems comprise large polymeric structures made up of building blocks or components (monomers) which are themselves made up of elements. Examples of large polymeric structures found in living systems include proteins made up of amino acids, DNA and RNA made up of nucleic acids, complex carbohydrates made up of sugars, and lipids which can include fatty acids. The ability to measure the formation and degradation of polymers within cells or in cell-free systems is integral to understanding regulatory processes controlling cell proliferation and death, and the nature of chemical reactions. The biosynthesis and degradation of a polymer is particularly important to an understanding of various disease processes, development of an organism, cellular differentiation, tissue remodeling, and the like.
Existing methods used for determining polymer formation and degradation within cells or organisms are often referred to as xe2x80x9cmetabolic labelingxe2x80x9d techniques. By far the most corrimon current technique for the in vitro metabolic labeling of cells or tissues in culture uses 35S-methionine as a probe to measure the formation or degradation of cellular proteins. The technique of using radioactively-labeled amino acids as metabolic probes dates back to the late 1940""s. (Tarver, et al., J. Biol. Chem. 167:387-394 (1947)).
Although the technique is widely used for in vitro determinations of protein synthesis, there are major disadvantages to using radioactive amino acids or other exogenous (where the probe is not a usual constituent of the polymer being studied) radioactive probes in metabolic studies. These disadvantages include: 1) the danger to personnel using ionizing radiation, as well as the necessity to acquire permits for the purchase, storage, and disposal of radioactive materials: 2) the need to collect all of the protein synthesized to permit quantitative determinations since the method measures absolute levels of radioactive signal: 3) exogenous probe often alters the physical properties of the polymer being studied; 4) many elements exist for which there are no known radioactive species; and 5) use of radioactive probes is generally limited to in vitro systems or animal models since the concentration of probe needed for human studies usually exceeds legislated xe2x80x9csafe-dosexe2x80x9d standards.
These difficulties in the use of radioactive probes can be largely overcome as provided by the present invention by the use of a probe containing a stable isotope.
That most elements are mixtures of a number of stable isotopes has been known for over 60 years, and stable isotopes have been used as probes for metabolic studies within humans. The use of these probes was initially limited by their high cost and limited availability. Over the last few decades, many techniques for the production of stable isotope probes have been developed, and highly purified probes containing stable isotopes are commercially available. Further, instruments for the analysis of isotopic peaks (mass spectrometers) are now readily available to most workers in the field.
Use of a stable isotope for analyzing incorporation of a probe into biopolymers during in vivo metabolic studies has several problems. These problems include but are not limited to: 1) the need to breakdown the biopolymer of interest into smaller components (often amino acids); 2) the need to chemically derivatize the components followed by separating the components within their classes (often by gas chromatography); and 3) analyzing the mass of each component using a mass spectrometer. (Halliday and Read, Proc. Nutr. Soc. 40:321-334 (1981)).
For each polymer of interest, the steps of separating the components within their classes and analyzing the mass of the components takes between 30 minutes to 1 hour. This greatly restricts the number of different polymers that can be analyzed on any one day. In addition, during the derivatization step, chemicals are added to the components, changing both the mass of the components and the isotope peak ratios in non-trivial ways (Lee et al., Biol. Mass Spectr. 20:451-458 (1991); Smith and Rennie, Biol. Clin. Endocrin. Metab. 10:469-495 (1996)). As stated by leaders in the field, xe2x80x9cThe technique is not without its disadvantages: the amino acid derivatization and quality control procedures are laborious and time-consuming; special instrumentation is required for precise GC-MS measurements; and a large number of time points are needed to accurately define the protein kinetics following a single-dose tracer administration.xe2x80x9d Patterson, B. W., et al., J. Lipid Res. 3:1063-1072 (1991); see also, Goshe and Anderson, Anal. Biochem. 231:382-392 (1995)).
Despite the limitations inherent in the use and analysis of stable isotopes. widespread acceptance of gas chromatography and mass spectrometry have hindered the development of faster and simpler forms of analysis. As recently stated in a review of the field (Bier. Eur. J. Pediatr. 156(1):S2-S8 (1997)), xe2x80x9cThe worldwide acceptance and primacy of gas-chromatography-mass spectrometry (GCMS) as the preferred analytical tool for identification of abnormal metabolites . . . confirms the general nature of this method, supports the position that its limitations are relatively small in number, and attests to the nearly absolute specificity of metabolite identification using this methodxe2x80x9d.
The scientific and medical community""s understanding of physiological processes is currently limited by the speed with which quantitative data can be collected, and the method by which the data is stored and processed in an easily retrievable fashion. Cobelli aptly summarizes this dilemma: xe2x80x9cThe principal difficulty attached to the mathematical analysis of physilogical and medical systems stems from the mismatch between the complexity of the processes in question and the limited data available from such systems.xe2x80x9d Cobelli, et al., Am. J. Physiol. 246:R259-R266, 1984. Thus, there remains a need in the art to develop high-throughput techniques for measuring polymer synthesis in these complex systems. Quite surprisingly, the present invention fulfills this and other related needs.
The present invention provides a non-radioactive technique for determining polymer formation or degradation, rapid processing and measurement of a large number of different polymers. In one aspect, the method includes adding a mass isotopically labeled component of a polymer (probe) to a system in which the unlabeled component of the same type as the probe has been depleted. Depleting the cellular pool of unlabeled component prior to adding the labeled probe increases the likelihood that during polymer formation, the labeled probe is incorporated into the new polymer. Over a period of time, the mass isotopically labeled probe will be incorporated into the new polymer formed, and the total pool of that polymer is the sum of the polymer. present prior to adding the probe and newly formed polymer which has incorporated the probe. The polymer of interest is isolated after a desired period of time in the presence of the probe. The isolated polymer can be cleaved into smaller fragments and the mass of the fragment and all isotopic peaks measured using an analytical instrument such as a mass spectrometer. For each fragment, the relative abundance of the different mass peaks from samples containing probe is compared to the mass peaks from samples where probe is absent. The ratios of fragment polymer mass peaks from samples treated with or without the probe are compared mathematically, and the relative proportion of polymer synthesized is determined. In one embodiment, the rate at which the polymer is synthesized is determined by measuring the relative amount of polymer formed at two or more time points, and dividing the difference in polymer synthesized at the different time points by the time interval.
The present invention also provides a method for storing experimental data within a searchable database to determine the identity of an individual polymer within a complex of several different polymers. Initially, an individual polymer can be separated from the complex of polymer, if desired, cleaved into smaller fragments, and the mass of the polymer or resultant fragments determined by an analytical instrument such as a mass spectrometer. For each parent polymer, a specific set of fragments having different masses is generated during the fragmentation procedure, but only a subset of these fragments is found within the mass spectra from the analytical instrument. The set of fragments of specific sizes that is measured by the analytical instrument can be used as a xe2x80x9cfingerprintxe2x80x9d to identify the parent polymer from which the fragments are derived. The database contains xe2x80x9cfingerprintsxe2x80x9d for a large number of different polymers, and is used to decipher the individual components of a complex comprising a number of polymers.
The process of measuring the relative amount and rate of polymer formation within cells or during a chemical reaction includes adding a labeled monomer probe containing one or more molecules of a stable isotope of an element within the structure of the monomer to cells growing in tissue culture or to a chemical reaction outside of a cell. After the desired incubation time in the presence of the monomer probe, a sample is taken and the chemical reaction is stopped. Products of the reaction are isolated and the relative abundance of the monoisotopic and isotopomeric peaks of the polymer produced are measured and compared to the relative abundance of the monoisotopic and all isotopomeric peaks of the polymer produced from a similar chemical reaction where no labeled monomer probe has been added. In the case of living cells, the cells are washed free of unincorporated monomer probe and solubilized to free the individual biopolymer, i.e., proteins, lipids, nucleic acids, complex carbohydrates, and the like. The resultant cellular polymers can then be separated and isolated by common techniques, and if desired, further fragmented enzymatically and the relative abundance of the monoisotopic and all isotopomeric peaks of the polymer or fragments thereof measured. The measured abundance for a test sample is then compared to the relative abundance of the monoisotopic and isotopomeric peaks of polymer isolated from solubilized cells treated in the same way except for the addition of monomer probe.
To determine the relative amount of polymer formed during a desired incubation time, a mathematical algorithm (Equation (1)) is applied to the comparison of the relative abundance of the monoisotopic and isotopomeric peaks from whole or fragmented polymers in the presence and absence of the probe.                               (                      1            -                                                            ∑                  C                                ⁢                                  xe2x80x83                                                                              ∑                  S                                ⁢                                  xe2x80x83                                                              )                xc3x97        100                            (        1        )            
To determine the rate of polymer synthesized or degraded, the relative amount of polymer is measured over two or more time-points and is equal to the slope of the line represented by the percent abundance of labeled polymer per unit time. When samples are taken at two time points the rate of synthesis or degradation is equal to the percent of labeled polymer at time 2 divided by the percent of labeled polymer at time 1.
The present invention also provides a database comprising mass spectra obtained for the polymers and polymer fragments in relation to particular descriptors. The mass spectra define a polymer or fragment thereof by the quantity of mass isotopically labeled monomer over a specified time period. A mass spectra for one or more polymers or a fragment of the polymers is used to characterize a particular protein found in a cell or tissue at a particular stage of development, differentiation, or physiological state.
In practicing the methods of the present invention to determine the rate of polymer synthesis or degradation, the data obtained for each polymer and polymer fragment analyzed for a particular population of cells can be entered into a database with associated descriptors. The descriptors include such characteristics as the species of organism, cell type, tissue type, state of differentiation, polymer class, method of separation of polymer class into separate parent polymers, method of fragmentation of parent polymer, and the like. Having established a database, an unknown cell, tissue sample or polymer is labeled with a stable isotopic monomer probe and the abundance of the labeled probe at certain time points determined. The rate of incorporation of the labeled probe and/or the xe2x80x9cfingerprintxe2x80x9d of fragments obtained for the parent polymer by a particular method is compared to the database, and one or more matches within the database can then be determined to identify the organism, cell, tissue type, polymer, etc. The xe2x80x9cfingerprintxe2x80x9d can also be compared to private or public databases comprising amino acid or nucleic acid sequence information, for example, or a monomer sequence can be determined for the isolated parent polymer or fragment thereof to identify the parent polymer. The data obtained for the unknown sample can be added to the database with all known descriptors to increase the size of the database.